The present invention relates to a method for obtaining a monocotyledon plant containing a gene of interest free of foreign ancillary sequence.
Plant transgenesis comprises introducing into a plant one or more genes originating from various organisms (bacteria, viruses, insects, plants), with the aim of providing it with novel characteristics and of improving certain agronomic or dietary qualities. The great diversity of genes, combined with the development of conventional techniques for genetic transformation, has resulted in the creation of novel plant varieties. In some cases, farming practices can be facilitated and yields increased by virtue of the introduction of characteristics which confer resistance to a herbicide, to pathogens or to various forms of stress. In other cases, the nutritive value of the plant and the content of certain essential compounds can be enhanced. However, the integration of genes into a genome during a process of transgenesis occurs with very low frequency. For this reason, in most cases, either a selection marker gene, which gives a selective advantage to the transformed cells, or a phenotypic marker, which allows those skilled in the art to identify the transformed cells among the others is genetically linked to the genes of interest.
Selection marker genes, such as genes for resistance to antibiotics or to herbicides, are essential for detecting the transformation events. However, they remain in the plant and, consequently, can also be detected in the form of DNA or of proteins in certain derived products, whereas generally they do not provide any added value to the transformed plant obtained. The presence of these genes, and in particular the genes for resistance to antibiotics and herbicides, is today at the center of numerous debates regarding genetically modified organisms (Flavell et al., 1992; Chesson et al., 2000).
It is therefore necessary to develop systems which make it possible to eliminate these selection genes, once the transformants have been isolated by virtue of the selective agent and/or by molecular analyses. Several more or less complex methods of elimination have been studied, such as:
cotransformation, which consists in introducing two T-DNAs (transfer DNAs) into the organism, the first with the gene of interest and the other with the selection gene. This method is used rather for species with a short reproductive cycle. Transformants carrying the transgene of interest but having lost the selection gene are then selected in the descendants by segregation;
systems which exploit recombination at specific sites, such as the cre/lox system of the P1 bacteriophage or the yeast FLP/FRT system (FliPase recombinase; Lyzrik et al., 1997) used for species with a long reproductive cycle (maize, shrubs); however, these systems remain complex and laborious;
elimination systems which exploit the properties of mobile genes present in many genomes, such as retroelements and transposons, and in particular the maize Ac/Ds system used in many species.
Transposable elements in general, including in particular the Ac/Ds system, can excise themselves and reinsert into the genome during cell development. These elements may also not find a recipient site into which they can insert themselves, and are then degraded by nucleases. Transposons are mainly used for cloning and tagging certain genes with visible expression phenotypes (Dellaporta et al., 1988), but they can also be used to mobilize, excise and eliminate genes (PCT/US91/04679).
A system using maize Ac/Ds and allowing the elimination of selection marker genes in the tomato, a species which does not naturally possess any Ac or Ds element, has been described by Yoder et al. (1993).
However, this system, which has been tried and tested in heterologous plants of the dicotyledon type (tomato, garden arabis, tobacco), appeared, up until now, difficult to exploit in monocotyledons, in particular maize, a species in which Ac/Ds is naturally present. Specifically, the presence of endogenous elements makes it difficult to control and exploit this system.